An efficient stress-free strategy to displace stable bacterial plasmids

Abstract

A key stage in determining the phenotype(s) conferred by a plasmid is its displacement, or 'curing,' to create a plasmid-free strain. However, many plasmids are very stable, not only because they contain multiple replicons, but also because they can encode post-segregational killing systems that reduce the viability of plasmid-free segregants. We have developed an efficient curing strategy that involves combining key regions of the replicons and the post-segregational killing loci into an unstable cloning vector carrying sacB, which confers sensitivity to sucrose. Targeting plasmids of both the F family of Escherichia coli and the broad-host-range IncP-1 family, we demonstrated displacement of susceptible resident plasmids from all clones tested. Growth on sucrose allowed the isolation of many clones without either plasmid. This strategy is highly efficient and avoids the stress of inducing and surviving the effects of post-segregational killing systems or other lethal gene products.

Curing vector for IncI1 plasmids and its use to provide evidence for a metabolic burden of IncI1 CTX-M-1 plasmid pIFM3791 on Klebsiella pneumoniae

“…In the first instance, the RNA gene, RNAI, which controls the copy number in IncI1 plasmids by translational regulation of replication initiator repZ (Praszkier & Pittard, 2005), was amplified by PCR and cloned into suicide vector pIFM26 (see supplementary methods). This vector is not stable in the absence of selection, which, together with the presence of negative selection marker sacB (confers sucrose sensitivity to Gramnegative organisms), would enable recovery of vector-free clones by growth on antibiotic-free, sucrose-containing media (Hale et al, 2010).…”

“…Thus, for this study we opted for the use of incompatibility-based curing following the observation that two plasmids bearing the same replicon cannot be stably maintained in a bacterium (Couturier et al, 1988). Plasmid incompatibility-based curing has been successfully employed for curing of plasmids (Hale et al, 2010;Tatsuno et al, 2001), but not as yet for IncI1 plasmids. Here we describe the construction of a curing tool for IncI1 CTX-M plasmids and then the phenotypic analyses of plasmid-free and plasmid-carrying strains.…”

Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

“…The pCRISPR- SacB - gDNA plasmid derives from a pCRISPR plasmid [9], where the kanamycin resistance gene ( Km R ) is fused to the sacB gene encoding the Bacillus subtilis levansucrase. SacB is toxic in E. coli if grown in media containing 5% sucrose [21–24] and, as previously demonstrated by Hale and co-workers, plasmid-borne sucrose toxicity can be exploited to cure high copy number plasmids [25]. We used this strategy to remove the pCRISPR- SacB - gDNA plasmid after the gene mutation has been introduced.…”

“…Elegant solutions have been proposed to get rid of high copy number plasmids [10, 18, 30]. We adopted the strategy proposed by Hale and co-workers [25] by introducing the suicide sacB gene downstream of the Km cassette into the pCRISPR plasmid, thereby generating the pCRISPR- SacB plasmid. E. coli strains carrying any pCRISPR- SacB plasmid derivative can survive in the presence of sucrose only if they rapidly lose the plasmid [21–24].…”

“…To get rid of our pCRISPR- SacB-gDNA we envisaged to exploit the toxic effect of the sacB gene when E. coli is grown in media containing high concentrations of sucrose. SacB fused to an antibiotic resistance marker (usually chloramphenicol) has been used in chromosomal gene knock-in/knock-out experiments [21–24] and, more recently, for plasmid curing [25]. Indeed, we found that by simply inoculating the mutant colonies in LB supplemented with 5% sucrose, bacterial cells rapidly lose the plasmid and, if necessary, the culture is ready to be used for an additional round of mutagenesis.…”